Design of customizable long linear DNA substrates with controlled end modifications for single-molecule studies

Authors

Stefan Mueller, University of WollongongFollow
Lisanne Maria Spenkelink, University of WollongongFollow
Antoine M. van Oijen, University of WollongongFollow
Jacob S. Lewis, University of WollongongFollow

RIS ID

140996

Publication Details

Mueller, S., Spenkelink, L., van Oijen, A. & Lewis, J. (2020). Design of customizable long linear DNA substrates with controlled end modifications for single-molecule studies. Analytical Biochemistry, 592

Abstract

2019 Many strategies have been developed to manipulate DNA molecules and investigate protein-DNA interactions with single-molecule resolution. Often, these require long DNA molecules with a length of several 10s of kb that are chemically modified at specific regions. This need has traditionally been met by commercially available DNA from bacteriophage λ. However, λ DNA does not allow for the generation of highly customizable substrates in a straightforward manner, an important factor when developing assays to study complex biochemical reactions. Here we present a generalizable method for the design and production of very long chemically modified DNA substrates derived from a single plasmid. We show the versatility of this design by demonstrating its application in studying DNA replication in vitro. We anticipate this strategy will be broadly useful in producing a range of long chemically modified DNA molecules required for a diverse range of single-molecule approaches.